This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We now are making good progress: ‘ We continue to screen potential genes for HLB control and are finding peptides that reduce disease symptoms and allow continued growth of infected trees. ‘ We have greatly improved our efficiency of screening . ‘ We are modifying the vector to express more than one anti-HLB gene. ‘ We are modifying the vector to allow addition of a second vector. ‘ We are preparing to put trees into the field for testing as soon as potential freezes are over. ‘ We continue to supply infected and healthy psyllids to the research community.
When dsRNA targeting either a psyllid cathepsin or a psyllid vacuolar ATPase gene are fed in artificial diets to the Asian citrus psyllid, an increase in psyllid mortality is realized. The oral uptake of ~300 bp dsRNA fragments matching the coding region to either psyllid Vacuolar ATPase or cathepsin can induce mortality in the Asian citrus psyllid. Comparisons were made to determine the optimal dsRNA size. Psyllids were fed either the ~300 bp dsRNAs directly or after processing to siRNAs with the Dicer enzyme. Results showed that the 300 bp dsRNAs induced greater mortality and than that observed with processed siRNAs. Furthermore, non-linear dose dependent toxicity of the ~300 bp dsRNAs suggesting complex interactions that have not yet been characterized with respect to dsRNA induced toxicity in insects.
Seed was collected from new crosses made the previous year to develop improved rootstocks and scions with tolerance to HLB and other improved traits. Selected hybrid seed was planted in the greenhouse. Promising SuperSour rootstock hybrids were identified and propagated for further testing. Cooperative work began with a commercial nursery to propagate the most promising SuperSour hybrids for large-scale commercial field trials. Fruit quality, yield, and tree size information were collected from 8 existing replicated rootstock and scion field trials. Performance data from field trials was summarized in a presentation at the Indian River Citrus Seminar. A field trial was planted in Indian River County to evaluate SuperSour selections in heavy flatwoods soil for tolerance of Phytophthora and Diaprepes weevil. A greenhouse study was completed to evaluate the tolerance of SuperSour rootstock hybrids to citrus tristeza virus. Data was collected from a replicated field trial in St. Lucie County to evaluate HLB tolerance of ten rootstocks exposed to natural infection in the field. Propagations from SuperSour rootstock hybrids were budded with ‘Hamlin’ scion to produce trees for disease testing and new rootstock field trials in the coming year. Field studies continued to assess the tolerance of different rootstocks to HLB with sweet orange scion under natural conditions, and work is beginning on a summary of those findings for publication. In coordinated research between this grant and the FCATP transgenic citrus grant to USDA, selected anti-microbial, insect resistance, and other genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with resistance to HLB and Citrus Bacterial Canker. Seed from the new USDA rootstocks US-812, US-897, US-802, and US-942 produced at the USDA Whitmore Farm was collected and provided to the Florida Citrus Nurserymen Association for commercial distribution. A study describing the tolerance of US-897 to HLB was published in the journal ‘HortScience’. In the study, both US-897 field trees naturally infected by HLB and greenhouse-grown trees that had been inoculated with Liberibacter through graft inoculation were observed to develop few or no symptoms of HLB, even though PCR clearly indicated the presence of Liberibacter infection. Typical symptoms of HLB found in other citrus, such as leaf blotchy mottle, shoot stunting and yellowing, and seed abortion were rare and not easily visible on US-897. It was noted that the HLB tolerance of US-897 probably is derived from its trifoliate orange parent. Experiments are planned to determine whether the tolerance to HLB found in US-897 results in increased tolerance for grafted trees with susceptible scions on US-897 rootstock. A greenhouse study is underway to compare the apparent HLB resistance of several different trifoliate hybrid rootstocks. Greenhouse studies continued to compare germplasm tolerance to HLB under carefully controlled conditions, and understand the impact of different factors on that tolerance. These studies will provide additional insights about how to engineer HLB resistant cultivars. Additional greenhouse and field studies are also underway to determine the most efficient methods to evaluate new citrus germplasm from crosses and transformation for resistance or tolerance to HLB. Greenhouse studies are underway to compare infection and symptom development in plants inoculated with Liberibacter by graft and Asian citrus psyllid.
1-The first objective of the second year was to build and start the operation of a plant growth room at the Citrus Research and Education Center in Florida (CREC). The growth room construction started on October 22nd 2010 and the projected finish date was February 11th 2011. There was a delay of a few weeks and the main contractor will finalize on March 25th, but the computer system contractor is still finishing the programming that will control the environmental conditions. We checked the growth room and it is working as expected however disposal of the waste stream will be a concern when the growth room is in full operation since the water that we will dispose needs to be collected in an external tank and test by the county to guarantee that we are not disposing contaminants that can affect the environment. We already started furnishing and placing equipment inside the building. Because of the delay in the construction, the growth room is not in fully operation yet. We believe this will take at least one additional month. The cost of the construction was higher than the original budget plan; the extra funding was provided by CREC and IFAS facilities as agreed initially. 2- A full time technician with nursery experience was hired after several months of searching. The process of hiring was slow. A first candidate was hired and the offer of employment was rejected due to a low salary offer. A second candidate was found but he quit two months after hiring. We hired a third technician with limited experience and he will start the first week of April. It seems like salary will be an important issue in the future to recruit and retain personal. 3- Training of the manager Dr. Zapata was completed at the IVIA under the supervision of Dr. Pena. It was emphasized during the training the improvement of transformation methods for more recalcitrant types, molecular analysis of the regenerants and plant material preparation at the greenhouse/growth room, including micrografting, phytosanitary treatments, fertilization and pruning. An annual schedule for completion of planting, transplanting, grafting and obtaining budsticks to transform was developed. 4- The Mature Transformation Laboratory was established, using an existing laboratory located at CREC. Two technicians with limited experience were hired and are currently being trained in the first tissue culture techniques, including culture media preparation, grafting, micrografting, explant preparation, culture and regeneration, etc. 5-Our selected varieties Hamlin 1-4-1, Pineapple S-F-60-3 and Valencia S-SPB-1-14-19 were subjected to cleaning through shoot tip-grafting at the Florida Department of Agriculture and Consumer Services (DOACS) with the help of Dr. Peggy Sieburth. They are kept at out lab and ready to be grafted on rootstocks when the growth room is fully operative. 6-Dr. Leandro Pena and his greenhouse and growth room manager Josep Peris made a visit of one week in March 2011 to supervise the last steps of the growth room construction before been finalized and suggested minor details to make the facility more reliable and helpful for operators. They also short-trained the tissue culture technicians in horticultural practices. They checked substrate, seed stock and nutrition issues with the manager Dr. Zapata.
The goal of this project is to transform the citrus and Arabidopsis NPR1 genes (CtNPR1 and AtNPR1), and the rice XIN31 gene into citrus, and to evaluate their resistance to both citrus canker (caused by Xanthomonas axonopodis pv. citri (Xac)) and greening diseases. The first year objectives include: (1) Molecular characterization of the transgenic plants; (2) Inoculation of the transgenic plants with Xac; (3) Inoculation of the transgenic plants with the HLB pathogen, and monitoring of the bacterium in planta with quantitative PCR; (4) Transformation of SUC2::NPR1 into citrus; (5) Plant maintenance. We have identified three transgenic lines overexpressing CtNPR1. These NPR1 overexpression lines were inoculated with 105 cfu/ml of Xac306 and the results showed high levels of resistance from the NPR1 overexpression lines, but not from the control plants. We also conducted growth curve analyses. Nineteen days after inoculation, the bacterial population in one of the NPR1 overexpression lines is 10,000 fold lower than that in the control plants. These results demonstrate that overexpression of CtNPR1 confers resistance to canker disease. We also graft-inoculated the NPR1 overexpression lines with greening to determine whether NPR1 is functional in greening resistance. We are in the process of monitoring Candidatus Liberibacter asiaticus populations in the inoculated plants using quantitative PCR. Five transgenic lines containing the SUC2::CtNPR1 construct, in which CtNPR1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene, have been generated. This construct may increase the expression of CtNPR1 in citrus phloem thereby maximizing the opportunity for resistance to greening. In a few weeks when the plants produce enough leaf tissue, we will perform Northern blot analyses to monitor the levels of CtNPR1 transcripts. The transgenic plants with high levels of CtNPR1 will be propagated by grafting and inoculated with greening. Finally, we have initiated microarray analyses of the CtNPR1 plants in response to Xac inoculations. The results will be reported soon. To continue our research, we request funds for the third year to achieve the following goals as originally proposed: (1) Characterization of the CtNPR1 transgenic plants inoculated with the HLB pathogen; (2) Molecular characterization of the SUC2::CtNPR1 plants; (3) inoculation of the SUC2::CtNPR1 plants with the HLB pathogen; (4) Examination of changes in hormone (abscisic acid, auxin, jasmonic acids and salicylic acids) levels in the CtNPR1 plants infected with Xac; (5) Plant maintenance.
This research project is directed towards controlling psyllids using biologically-based control strategies that employ the use of RNAi technology against key biological control pathways, peptide hormones and protein inhibitors that, if expressed in transgenic citrus, would enhance plant resistance to psyllid feeding. Both protein-based and RNAi strategies were tested by feeding psyllids artificial diets. To support the artificial diet assays, we optimized the diet composition by adding an antimicrobial agent to eliminate fungal growth that is introduced by the psyllids during the assay period. Using this approach we identified suitable buffers and optimal diet pH during the feeding period. In separate experiments, Tryspin Modulating Oostatic factor (TMOF), a mosquito decapeptide hormone, and cysteine protease inhibitor (CPI) from the Asian Citrus psayllids that was identified in our laboratory were added to artificial diets on which psyllids were allowed to feed. After 10 days of feeding, 100% mortality was observed in psyllids feeding on diets containing TMOF or CPI, whereas, 40% mortality was found in psyllids feeding on the control diets. CPI fed psyllids caused a significant higher mortality than the controls after 7 days of feeding. Based on these observations a collaborative project with Dr. Bill Dawson’s laboratory (Univ. of Florida, IFAS, CREC, Lake Alfred, FL) was initiated to use a Citrus tristeza virus (CTV) expression vector to produce TMOF and CPI in the in the citrus phloem. Clones containing the sequences of mosquito TMOF and CPI (cathepsin protease inhibitor) (both shown to be effective against psyllids) were provided to Dr. Dawson’s laboratory. Once CTV vectors are constructed and used to infect citrus plants, Dr. Dawson’s laboratory will provide us with the plants to evaluate the effect on psyllids while feeding on these plants. In parallel to these studies, we synthesized dsRNA molecules targeting 11 different psyllids essential genes encoding three different classes of proteins (alpha-tubuliln, V-ATPase, and Cathepsins). Initial feeding studies with alpha-tubulin dsRNA and V-ATPase dsRNA caused ~60% psyllids mortality as compared to only ~30% mortality for psyllids fed a control diet containing an equal amount of dsRNA not specific to the psyllid. Using this information, we have initiated experiments to produce citrus plants that express these dsRNAs in citrus phloem cells. A collaborative project with Dr. Bill Dawson’s laboratory (Univ. of Florida, IFAS, CREC, Lake Alfred, FL) was initiated to use a Citrus Tristeza Virus (CTV) expression vector to produce the psyllid dsRNAs in the citrus phloem. Clones containing the sequences of a psyllid Vacuolar ATPase and Cathepsin protease (both shown to be effective dsRNA targets) were provided to Dr. Dawson’s laboratory. Once CTV vectors are constructed and used to infect citrus plants, Dr. Dawson’s laboratory will provide us the plants to evaluate the effect on psyllids while feeding on these plants. We have also designed expression vectors for the production of transgenic plants expressing the psyllid dsRNAs through Agrobacterium-mediated plant transformation. These will be used to initiate the production of transgenic citrus plants that constitutively produce these dsRNAs in the phloem.
New lemon selections suitable for the California desert climate are needed to diversify production. Desert lemons occupy an important early-season market niche, which could be lost to international competitors. Lemons are also an important source of fruit for packinghouses located in other areas of the state. This ongoing project, which addresses the CRB ‘Production’ priority for New Variety Development, was designed to evaluate twelve lemon selections under desert conditions. The immediate objectives of the project are to provide the industry with information on the tree growth, yield, packout, and fruit quality characteristics for the lemon selections in the California desert. These include: ‘Allen Eureka’, ‘Variegated Pink-Fleshed Eureka’, ‘Corona Foothills’ (a bud sport of ‘Villafranca’), ‘Limoneira 8A Lisbon’, ‘Walker Lisbon’, ‘Femminello Santa Teresa’, ‘Interdonato’, ‘Limonero Fino 49′, Limonero Fino 95’, ‘Messina’, ‘Seedless’ lemon and ‘Yen Ben’. There are 20 trees of each variety, grouped into five replications of four trees each. All are budded to Citrus macrophylla rootstock. Leaves were collected for nutrient analysis for all 12 varieties in September, 2010; these are currently being processed and analyzed. The first harvest for 2010 took place on November 2, 2010, using professional harvesters from a local packinghouse. We collected yield from each group of four trees by counting the numbers of whole and fractional picking sacks harvested from each group. Also, we collected fruit packout and exterior fruit quality from each variety using a portable fruit line. For the first harvest, ‘Corona Foothills’, Limonero Fino 49′ and ‘Walker Lisbon’ had the greatest yields ranging from 200 to 250 lbs. per tree, while ‘Seedless’ lemon, ‘Variegated Pink-Fleshed Eureka’ and ‘Yen Ben’ had the least yield of less than 75 lbs. per tree With regards to fruit packout, ‘Messina’ had the largest size, peaking on sizes 75 and 95, while ‘Corona Foothills’, Interdonato’, ‘Limonero Fino 49’ and ‘Limonero Fino 95’ peaked on sizes 95 and 115. ‘Variegated Pink-Fleshed Eureka’ and ‘Yen Ben’ had the smallest sized fruit, peaking on size 140. There was little effect of selection upon fruit exterior quality. Samples of 20 fruit per group of four trees were collected for determination of interior fruit quality, including peel thickness, peel smoothness, percentage juice, juice pH and TSS/TA ratio. These data have not yet been analyzed. Several growers attended a Field day and saw the trees first-hand at the experimental block on September 28, 2010.
Two trees have been found growing in HLB-ravaged orchards in Guangdong and one other in Guangxi province, that appeared to be free of HLB symptoms, while all other trees planted at the same time were either dead or declining, and replants likewise were afflicted. The trees from Guangdong were propagated at the Guangdong Institute of Fruit Tree Research facilities, and are now planted in their research field to assess their reaction to natural inoculation with HLB. We visited these trees in October 2010 and observed no HLB symptoms, though surrounding trees showed obvious signs of disease. The tree in Guangxi that was transplanted to a protected location at the Guangxi Citrus Research Institute, was used to make several propagations, and these were deliberately inoculated by grafting infected material. These propagated trees have expressed symptoms of HLB now. They apparently are not resistant, but the question remains as to why the original source tree was not infected, and still appears symptom free. We visited with a citrus extension specialist from the Fujian Provincial Academy of Agricultural Sciences, Mr. Li, Jian, also in October 2010, to expand further our search for survivors, and to continue to learn about Chinese citrus industry adjustments in response to HLB. We visited the Pinghe County Guanximiyou (Chinese honey pummelo) production area, where we saw HLB being managed through good psyllid control, good nutrition and subsequent tree health, and the natural tolerance of this pummelo variety. We returned to specific orchards we visited previously in Guangdong and Guangxi. These were in very healthy condition in 2008, despite widespread infection of neighboring plots. The orchard in Guangdong was devastated with HLB, and we met the grower again; we were informed that fruit prices for his variety were very low and he simply stopped taking care of his trees. Orchards revisited in Guangxi were a different matter. These orchards have not only maintained their healthy condition, but they were substantially more productive and healthier in appearance than two years earlier. This confirms the utility and effectiveness of the management strategy being employed under direction of entomologist Deng of the Guangxi Citrus Research Institute. We interviewed growers, pathologists, horticulturists, and entomologists associated with these healthy orchards. The key elements outlined to us were critically timed pesticide applications, use of pathogen-free planting materials, and maintenance of tree health through good nutrition.
The objectives of this project are to develop, test and evaluate citrus rootstocks for disease and pest tolerance, and to select stocks that impart to the scion high yield, superior fruit quality, acceptable fruit size, and other essential traits. Activities involve production on new potential rootstocks by hybridization, screening these for valuable traits, and analysis of performance in replicated field trials. This report summarizes accomplishments from November 2010 to February 2011. We completed data collection for a rootstock trial with Moro blood orange scion. Yield, packline, and fruit quality analysis were completed for the Fukumoto rootstock trial at Lindcove. A trial of satsumas on 4 rootstocks was also harvested as planned. We continue to propagate trees of Washington navel on 28 rootstocks for a new trial to evaluate rootstock effects on early bearing and responses to high intensity management. This trial should be planted during summer of 2011. Seedlings for planned trials of Clementine and DaisySL mandarins are being grown at Lindcove, but these did not grow well after transplanting and we may have to start this trial again. Seeds were collected from standard rootstocks and new hybrids to be used in future rootstock trials and for future iron chlorosis and salinity tolerance screening trials. A Phytophthora citrophthora root rot resistance trial was planted in January.
TThe objectives of this project are to develop new mandarin, orange, lemon and grapefruit-type cultivars suitable for California conditions. The major approaches being used are hybridization-selection for mandarins and grapefruit, and mutation induction by gamma irradiation to obtain seedless forms of existing mandarins, oranges, and lemons. Promising selections are evaluated in field trials for tree size, yield, fruit quality, and disease susceptibility. This progress report summarizes accomplishments from November 2010 to February 2011. Hybridization during the 2010 season was quite successful and we have been harvesting and initiating culture of large numbers of new hybrid seed. No new irradiation has been conducted yet this year. Most work this year has involved evaluation of hybrids and selections from mutation breeding. Evaluation of new hybrid populations and trees grown from mutagen-treated buds is on schedule, with hundreds of trees evaluated to date. These include a population to be used to map genes influencing fruit quality and other traits. In the 6 replicated trials, detailed fruit quality studies are being conducted on DaisySL, FairchildLS, Nova IR10, KinnowLS, Encore IR6, 3 hybrid mandarins, and one grapefruit type, with Clementine and Tango as early and late season comparators. KinnowLS was released for propagation in California in late January 2010.
Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. A journal manuscript was submitted on research showing that use of the antioxidant lipoic acid significantly improves transformation efficiency in Mexican lime; experiments to test this with commercial sweet oranges are underway. We continued with experiments to test the effects of various antibiotics / metabolites / herbicide on the transformation efficiency, including: kanamycin, hygromycin, mannose and phosphinothricin. ‘New publications supported by this grant:1. Dutt, M., D.H. Lee and J.W. Grosser. 2010. Bifunctional selection-reporter systems for genetic transformation of citrus: mannose and kanamycin based systems. In Vitro Cellular & Developmental Biology-Plant 46:467-476; 2.Orbovic, V., M. Dutt and J.W. Grosser. 2010. Seasonal effects of seed age on regeneration potential and transformation success rate in three citrus cultivars. Scientia Horticulturae 127: 262-266 Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Working with Mr. Orie Lee and a commercial harvesting company (w/ Frank Rogers), a plan to collect meaningful yield and fruit quality from the St. Helena project was developed – with harvest expected later this month. Approximately 10 acres of trees planted 2.8 years ago include a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks. The majority of trees have a significant yield and the trial is showing significant rootstock affects on precocious bearing and early fruit quality – the best selections from this trial will be ideal candidates for testing with juvenile transgenics. Also of interest is the cultural program being used at the St. Helena project that mimics OHS principals but with reduced input. The trees have been grown with a UF research slow-release fertilizer mix (in cooperation with Harrell’s Fertilizer) and daily irrigation. Two trees were confirmed with HLB the first year; but even with bad neighbors, there has been no detected additional spread of HLB during the past year. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious OLL sweet oranges (a group of clones with Rhode Red quality that show high solids in young trees) were regenerated and successfully micrografted for further study of early flowering and transgene expression. Approximately 25 transgenic sweet orange trees from OLL selections were produced containing four different gene constructs. Progress was also made transforming OLL clones with the alternative embryogenic culture transformation system, as numerous transformed somatic embryos have been recovered. Progress was also made in the regeneration and characterization of plants containing the FDT transgenes for early flowering.
Objective 1: A comparative study of two susceptible hosts, Duncan grapefruit (DG, C. paradisi), and Rough lemon (RL, C. jambhiri) and two resistant species of kumquat (Fortunella spp.), ‘Meiwa’ and ‘Nagami has been conducted to evaluate the basis for resistance to Xanthomonas citri subsp. citri (Xcc). The type of resistance occurring in kumquats is a hypersensitive response (HR) that develops within 48-72 h. This is based on the phenotype of the lesion, histological changes at the cellular level of infected tissue and the early expression of genes related to programmed cell death (PCD). In kumquats but not in DG and RL, several HR-related genes are expressed at 4 h post-inoculation (pi) with Xcc at 108 cfu/ml. The genes that are over-expressed are lipoxygenase, glutathione transferase, metacapcase, acid chitinase and peroxidases that have been linked with PCD. Later at 24 h, additional genes related to plant defense like PR-2 (betaglucanase) are highly expressed in kumquats but less so in DG and RL and their activity continues to increase up to 48 h pi. Different sets of genes are expressed in susceptible DG an RL related to the host pathogen interaction. The gene expression analyses have been extended to include comparisons of susceptible RL 8166 with the RL cybrid (RL + Valencia orange as the cytoplasm donor). Validation of the inheritance of resistance for the RL 8166 cybrid and newly developed cybrids of susceptible Ruby red grapefruit (RG) with Valencia orange (VO) is underway. The resistance inherited from VO is not HR (qualitative) but instead a degree of quantitative resistance compared to highly susceptible RG. Evidence for this is based on an intermediate lesion phenotype for cybrids in vitro and in-planta. In contrast to development of callus in susceptible RL and RG, the inoculated area develops more necrosis by 10 days pi. Xcc populations in the cybrids plateau at a level below populations in RL or RG. The lesion type is a mixture of necrotic and callus tissue that indicates that some cell death occurs and arrests the proliferation of Xcc. Expression of HR- and host pathogen interaction related genes in the RL cybrid is intermediate between kumquats and RL. The different pattern of gene expression suggest an interaction between the parent nuclear genes with the heterologous mitochondria and chloroplast from the cytoplasmic donor (VO). Field trials with several Ruby red grapefruit cybrids planted in canker-affected locations on the east coast continue to show less foliar disease incidence than the adjacent Red grapefruit trees. The production of cybrids lines using a new callus line of Meiwa kumquat as the cytoplasmic donor is underway in the Grosser lab. A new device for precise inoculation of Xcc bacteria into citrus leaves has been published and the prototype of the newly designed instrument is currently being used in several projects related to citrus canker.
Preliminary work to explore optimal parameters for genetic transformation of Murraya has been carried out, including assessments of shoot sensitivity to the selection agent kanamycin using untransformed shoots, determinations of bacterial growth curves, and appropriate and effective antibiotic concentrations for bacterial selection. Using the recently optimized protocol for organogenic shoot regeneration from appropriate seedling tissues, transformation experiments have been initiated using the plasmid pTLAB 21 harbored in Agrobacterium tumefaciens strain EHA 101. This vector contains the genes for kanamycin resistance as a selection agent and GFP (green fluorescent protein) as an easily observed marker. Various factors, including a range of OD values (cell density or concentration in liquid culture) of Agrobacterium cultures, the duration of explant incubation in bacterial cultures, duration of co-cultivation period, and the composition of co-cultivation and regeneration media were tested, to attempt establishment of an optimal and standardized transformation protocol. Optimal conditions for transformation using shoot tips and lateral buds, to develop an alternative method using a different tissue source should the organogenic approach prove too difficult or inefficient for transformation, were also explored. Regeneration of buds and some shoots has occurred from organogenic cultures of longitudinally cut seedling epicotyl segments, following these transformation experiments. Observations of the regenerating cultures have revealed several buds and shoots displaying green fluorescence, indicating successful genetic transformation. Their growth is being monitored, as well as the stability and uniformity of GFP expression over time, and further production of new transgenic events is underway.
Because of successful efforts to achieve acceptable seed germination rates and to produce more vigorous seedlings in vitro, higher quality seedling explant materials have been obtained. This accomplishment has enabled work to proceed to establish a suitable and efficient protocol for in vitro regeneration of Murraya, which can then be used for the various planned transformation experiments. Research was conducted to develop a suitable and robust organogenic shoot regeneration protocol, using various parts of germinated seedlings including hypocotyls (longitudinally cut vs. no cut), epicotyls (longitudinally cut vs. no cut), roots, leaves, and cotyledons. These explants were cultured on MS basal medium supplemented with various concentrations of BA, TDZ alone, or in combination with NAA. Two suitable and reasonably effective shoot regeneration protocols have been developed and optimized for transformation experiments (a manuscript is in preparation). Simultaneously, the capacity of shoot tips and lateral buds to undergo multiple shoot formation and multiplication in vitro has been examined. Shoot tips and lateral buds were excised directly from in vitro germinated seedlings and cultured on MS medium supplemented with BA at 0, 1, 3, and 6 mg/l. Newly formed shoots were subcultured on the same medium at 3 week intervals to determine optimal conditions for maximal shoot multiplication.
Quarterly report April 2011: Reserach to explore optimal various parameters for genetic transformation of Murraya was carried out, including assessments of shoot sensitivity to the selection agent kanamycin using untransformed shoots, determinations of bacterial growth curves, and appropriate and effective antibiotic concentrations for bacterial selection. Using the recently optimized protocol for organogenic shoot regeneration from appropriate seedling tissues, transformation experiments have been initiated using the plasmid pTLAB 21 harbored in Agrobacterium tumefaciens strain EHA 101. This vector contains the genes for kanamycin resistance as a selection agent and GFP (green fluorescent protein) as an easily observed marker. Various factors, including a range of OD values (cell density or concentration in liquid culture) of Agrobacterium cultures, the duration of explant incubation in bacterial cultures, duration of co-cultivation period, and the composition of co-cultivation and regeneration media were tested, and we established an optimal, standardized transformation protocol. Optimal conditions for transformation using shoot tips and lateral buds, to develop an alternative method using a different tissue source should the organogenic approach prove too difficult or inefficient for transformation, were also explored. Regeneration of buds and some shoots occurred from organogenic cultures of longitudinally cut seedling epicotyl segments, following these transformation experiments. Observations of the regenerating cultures revealed several buds and shoots displaying green fluorescence, indicating successful genetic transformation. Their growth was monitored, as well as the stability and uniformity of GFP expression over time. Nearly all of these transformation events proved to be either chimeric or transient, so further production of new transgenic events is being pursued.
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